PEGylated mutated Clostridium botulinum toxin

ABSTRACT

The invention relates to a modified botulinum toxin comprising a natural heavy chain and a modified light chain, characterized in that the modification of the light chain resides in that it comprises (i) an extension of the chain on its N-terminus which has the structure —(C) n -(tag) m -(X) l - in the direction from the N- to the C-terminal end, wherein
         C represents a cysteine residue,   tag represents any tag and   X represents the residue of any naturally occurring amino acid,   n represents an integer from 1 to 50,   m represents 0 or 1, and   l represents 0 or an integer from 1 to 50,
 
and in that (ii) at least one of the cysteine residues in the extension of the chain is coupled to at least one chain of PEG.

This application is a divisional application of U.S. Ser. No.12/282,601, filed Jan. 12, 2009 now U.S. Pat. No. 8,003,601, which is anational phase application under 35 U.S.C. §371 of InternationalApplication No. PCT/EP 2007/002296 filed Mar. 15, 2007, which claimspriority to European Patent Application No. EP 06 005 300.6 filed Mar.15, 2006. The entire text of each of the above-referenced disclosures isspecifically incorporated herein by reference without disclaimer.

The present invention relates to modified botulinum toxins (BoNT), whichhave enhanced stability and thus a prolonged therapeutic duration ofaction as compared to the corresponding native botulinum toxins.Furthermore, the present invention relates to pharmaceuticalcompositions comprising these modified botulinum toxins. Finally thepresent invention relates to nucleic acids, which encode these modifiedbotulinum toxins.

BACKGROUND OF THE INVENTION

Clostridium botulinum is an anaerobically growing, sporulatingbacterium, which produces a highly toxic protein. This so-calledbotulinum toxin is the cause of botulism, a food poisoning, whichwithout the use of intensive care measures can lead to the death ofbotulism patients. Seven serotypes are distinguished (type A-G, shortlytermed BoNT/A, BoNT/B, etc.) that have a similar amino acid sequence,but induce a different antibody response. The toxins (hereinafter alsoreferred to as neurotoxins and botulinum toxins) consist of twofunctional chains, the light (˜50 kDa) and the heavy chain (˜100 kDa),which are generated by proteolytic cleavage of the single-chainprecursor protein. Other strains do not possess the correspondingprotease, therefore the cleavage into the chains takes place in thegastrointestinal tract of the patients (e.g. by trypsin). In thedouble-chain form the subunits (i.e., the heavy and the light chain) areinterconnected via disulfide bridges (for example, in addition thereexists an intramolecular disulfide bridge in BoNT/A, i.e., between twocysteine residues of the heavy chain).

Under acidic conditions in vivo the pure neurotoxins do not exist infree form, but form complexes with other clostridial proteins, theso-called (Clostridium botulinum) toxin complexes. Different proteins,inter alia with hemagglutinating properties, are involved in thesecomplexes. The composition of the complexes is different betweenserotypes. The integration into the complex protects the neurotoxinduring gastrointestinal passage. These other clostridial proteins (thecomplexing and complex proteins, respectively) possibly play also a rolein the resorption of the neurotoxin. Thus, the incorporation in thecomplex causes the neurotoxin to be orally bioavailable and to thusconstitute a food poison. The target location of the neurotoxins is atthe motor endplate, where the muscle is activated by the nerve. Themotoneuron releases acetylcholine for activation of the muscle. Thisrelease is inhibited by botulinum toxin. The inhibitory effect takesplace in 3 sequential steps: binding, translocation, proteolysis. Theheavy chain of botulinum toxin binds highly specific to the motoneuronand is subsequently taken up into the nerve cell by endocytosis.Upstream of the binding domain, which is located at the C-terminal endof the heavy chain, there is the translocation domain in the N-terminalportion of the heavy chain, which transports or, rather, facilitates thetranslocation of the light chain into the cytosol by an as yet unknownmechanism. In the cytosol the light chain becomes active as a proteasecleaving highly specific so-called SNARE proteins. The proteolyticspecificity of the individual botulinum toxin types is summarized inTable 1. These SNARE proteins are responsible for the fusion of theacetylcholine-loaded secretory vesicles with the cell membrane of themotoneuron. The proteolytic cleavage of one of these SNARE proteinsinhibits the formation of a fusion complex and thus further release ofacetylcholine. The affected muscle is no longer activated. Previouslyhyperactive muscles become paralyzed.

TABLE 1 SNARE protein Botulinum Toxin Substrate of protease Cleavagesite in SNARE Type activity sequence from rat Type A SNAP 25 EANQ¹⁹⁷RATK Type B VAMP 2 GASQ⁷⁶ FETS Type C Syntaxin DTKK²⁵⁴ AVKY SNAP 25ANQR¹⁹⁸ ATK Type D VAMP 2 RDQK⁶¹ LSED Type E SNAP 25 QIDR¹⁸⁰ IMEK Type FVAMP 2 ERDQ⁶⁰ KLSE Type G VAMP 2 ETSA⁸³ AKLK

This mechanism of action is taken advantage of in the therapy of amultitude of muscle disorders and spasms, respectively, characterized byan uncontrolled release of acetylcholine (e.g., blepharospasm,torticollis, spasticity) Extremely low amounts of the neurotoxin (in thepg to ng range) are injected in the hyperactive muscle for therapy ofdystonia. The neurotoxin diffuses to the motor endplate and reaches thecytosol of the neuron to inhibit the acetylcholine release there. Themuscle is paralyzed after 1-2 days.

Various facial wrinkles are formed by cramping of muscles lying beneaththe skin, thus also through uncontrolled release of acetylcholine.Botulinum toxins find cosmetic utilization in this context: wrinkleswill be removed for about 3 months through injection of extremely lowamounts of botulinum toxin.

At present four preparations containing botulinum toxins have receiveddrug-regulatory approval: Botox® (Allergan), Xeomin® (Merz), Dysport®(Ipsen), and NeuroBloc® (Solstice Neurosciences). Botox®, Xeomin®, andDysport® are lyophilisates of botulinum toxin type A (as complex,neurotoxin and complex, respectively), Botox® and Xeomin® with 100 unitsper injection vial each, Dysport® with 500 units. NeuroBloc® containsbotulinum toxin type B (as complex) with 5,000 and 10,000 units,respectively, in liquid formulation.

Except for NeuroBloc the preparations are available as lyophylisates,which are reconstituted with physiological saline and are injected inthe respective muscles in matched doses depending on preparation andindication. The treated muscle will be paralyzed within 48 h. The effectlasts about 3 month, thereafter a further injection must be carried out,if the muscle should remain paralyzed further, i.e. the dystonia is tobe treated. Up to now it has not unambiguously elucidated, whichprocesses control the decrease of the effect. As long as the light chainis active as protease, the appropriate SNARE protein is cleaved (e.g.,SNAP 25 through the light chain of neurotoxin type A).

Accordingly, the fusion of the secretory vesicles with the plasmamembrane and thereby the release of acetylcholine will be inhibitedunder these conditions, the muscle remains paralyzed. If it werepossible to maintain the protease activity of the light chain for anextended time period in the cell, then the duration of action of anappropriate drug would be extended also

In contrast to many low molecular active substances active proteinsubstances are characterized by a significantly lower stability. Thehalf life (HL) of some active protein substances in the circulatingblood amounts to only a few minutes, so that the (therapeutic) durationof action is strongly restricted and injection must be repeated in shortintervals. The HL can be extended, if one is successful in protectingthe protein against degradation and elimination processes. Onetheoretically possible way exists especially for eukaryotic proteins ina higher glycosylation (more carbohydrate moieties) and in adapting thecarbohydrate structures to the structures of human glycoproteins,respectively. Another path that has been taken in a series of approvedactive substances is the coupling of the protein with polyethyleneglycol (PEG). PEG can be covalently bonded to the residues of variousamino acids, e.g., to lysine (amino function) or cysteine residues (SHfunction). PEG enhances the molecular weight of the protein withoutcreating immunogenic structures that induce the generation of antibodiesto the active substance. To the contrary: the PEGylation reduces theimmunogenicity of the active substance. The protein is eliminated moreslowly by the increase of the molecular weight and a significantincrease in HL is achieved. For maintaining a certain required serumlevel, the drug has to be injected less often.

PEGylated active protein substances are already processed in someapproved drugs (see Table 2). The employment of the partly smallproteins (e.g., interferon α 2a: Mr=19.3 kDa) in the original form, i.e.not modified, has shown that the proteins are very rapidly eliminatedfrom the serum. The PEGylation gave rise to a markedly increasedmolecular weight and thus to a substantially longer half life in theserum. Thus, for example, the serum half life for interferon α 2a is 9h; PEGylation with a 40 kDa PEG chain drastically increases themolecular weight and extends the half life from 9 to 72 h.

TABLE 2 Starting Trade name compound Coupling of PEG Pegasys interferonα 2a branched PEG-N-hydroxysuccinimide; Coupling to 4 lysine residuesNeulasta G-CSF PEG-aldehyde; Coupling to N-terminal methionine Peglutroninterferon α 2b Succinimidyl carbonate-PEG; Coupling to histidine andlysine residues Somavest growth hormone 4-6 PEG; antagonist Coupling tolysine residues and N- terminus Oncaspar AsparaginaseN-hydroxysuccinimide activated PEG

However the linkage with one or more PEG chains is subject torestrictions:

-   -   1. Preferably the PEG chain diminishes the biological activity        of the modified protein (in comparison with the unmodified        native protein) not at all or only slightly (in accordance with        the invention it is understood that slightly diminished        biological activity of the modified protein corresponds to at        least 20%, preferably to 30-40% or 50-70% or even to 75-95% of        the biological activity of the unmodified native protein). A        diminished activity is tolerable in many cases: e.g. the        antiviral activity of PEGylated interferon is 25-35% of the        non-PEGylated interferon a 2b. PEGylated interferon a 2a even        possesses only 1-7% of the activity of the non-PEGylated form.    -   2. As a multitude of therapeutically employed proteins deploy        their activity through the binding to a specific receptor,        preferably the PEGylation does not affect, or only slightly        affects, the interaction with the receptor (e.g., the        interaction can be affected directly by steric hindrance at the        binding domain or by alterations of the spatial structure of the        protein that have an effect on the binding domain and hence on        binding).    -   3. When the pharmacological effect of the therapeutic protein is        (also) mediated through an enzymatic activity (as for instance        with asparaginase), preferably the enzymatic activity is not, or        only slightly, reduced through the PEGylation.

Preferably the PEGylation of botulinum toxin accomplishes these threecriteria. At the same time the modification of the botulinum toxin withPEG preferably influences neither (a) the binding domain of the heavychain nor (b) the enzymatic activity of the light chain, i.e., the PEGchain preferably does not inhibit the interaction of the catalyticdomain from the light chain with the substrate (SNARE Protein). Incontrast to other proteases, that cleave short peptides, botulinumtoxins require longer peptides as substrates. For instance, a peptidewhich serves as a substrate for botulinum toxin type B preferably has asequence of about 40 amino acid residues of the SNARE protein VAMP 2.Peptides with shorter SNARE sequences will also be cleaved, but withsubstantially lower efficiency. The cleavage domain of the light chainof the botulinum toxin, which has a length comparable to the recognitionsequence of about 40 amino acid residues, is preferably not affected bythe PEG chain. Moreover, it has to be considered, that besides thecleavage domain responsible for the direct contact of the substrate(SNARE protein and peptide with the SNARE sequence of about 40 aminoacid residues, respectively) with the light chain, additional contactsites with sequences on the light chain located distantly to thecatalytic domain are needed for optimal activity of botulinum toxin. Ithas been demonstrated that five additional contact sites for itssubstrate SNAP 25 are localized on the light chain of botulinum toxintype A: 4 α exosites (AS 102-113, 310-321, 335-348, 351-358) and one βexosite (AS 242-259). Preferably, the contact is not or only marginallyimpeded through a conjugation of the light chain with PEG. Moreover, theC-terminal part of the heavy chain, the translocation domain, must beoperable, i.e., it must ensure that the light chain is transported fromthe endosomes into the cytosol. This transport process that isabsolutely necessary for the action can also be inhibited through thesteric hindrance of a PEGylated light chain especially as thetranslocation domain possibly forms a pore in the endosomal membranethrough which a “bulky” PEGylated light chain might not be channeledthrough.

Coupling of PEG to botulinum toxin is reported in a U.S. patentapplication (2002/0197278). The coupling serves to diminish theantigenicity and immunogenicity, respectively as well as to enhance themolecular weight for reducing the diffusion. For the selection of theappropriate sites (antigenic determinants) and amino acid residues,respectively, for the PEGylation, reference is being had to the paper ofBavari et al. (Vaccine 16: 1850-1856, 1998). In this paper sequences ofthe botulinum toxin heavy chain that induce neutralizing antibodies arepresented. In the aforementioned patent application it is only statedthat (1) the PEGylation should be carried out at, respectively close toone site or at, respectively, close to the sites, which act(s) as animportant epitope(s), but which are remote from the catalytic domain(i.e. remote from the light chain) and that (2) PEG may be conjugated tothe free terminal carboxy or amino groups or at the amino groups oflysine side chains. (3) As additional alternative for the insertion ofPEG into the toxin it is suggested to use the SH groups of naturallyoccurring or specially inserted cysteine residues; however, the paperadvises against this alternative (3), as disulfide bridges between theheavy and the light chain of the botulinum toxin play a role in thespatial configuration of the molecule. There is no example given thatdiscloses the structure of the PEGylated neurotoxin or that discloses onwhich amino acid residue(s) a PEG molecule of a certain length wasattached.

In a further patent application (WO 02/40506) relating to the change instability, the insertion, the modification or the removal of sites forthe in vivo glycosylation, in vivo phosphorylation and primarily the invivo myristoylation in the botulinum toxin are suggested in order tooptionally either enhance or decrease the stability of the botulinumtoxin. A whole series of potential modification sites are specifiedwhich are located at a significant distance to the N- and C-terminalends of the neurotoxin light chain. Additional sequences are to beinserted into the polypeptide chain, where carbohydrate chains orphosphate and myristoyl moieties, respectively, are coupled at the lightchain by cellular enzymes. Information regarding an accordingly modifiedneurotoxin or its preparation is however missing.

In a further U.S. patent application (2003/0027752) a peptide residuewith a so-called leucine motif (e.g., XEXXXLL) is inserted into theneurotoxin or into the light chain in order to increase the stability ofthe light chain within the nerve cell. The configuration of the lightchain with this motif ensures that it is localized in the vicinity ofits substrate at the membrane. Moreover, a so-called “tyrosine basedmotif” (YXXHy, Y=tyrosine, Hy=hydrophobic amino acid) is set forth that,after insertion in the light chain, is to enhance its persistency.Finally this patent application suggests a modified botulinum toxin typeA, in which the light chain is mutated (alanine to leucine at thepositions 427 and 428).

In view of the above described prior art it was an object of theinventors to provide an additional or precisely described form ofstabilization for any type of botulinum toxin preferably, however, fortype A, B, and C1. Along with this the object of the inventors was toprovide stable variants/analogs of the natural botulinum toxins which incomparison to the respective unmodified botulinum toxins have anincreased in vivo stability. This means, firstly, that the biologicalactivity (according to the invention biological activity is defined astotal activity comprising the enzymatic/catalytic activity of the lightchain as well as the required neurotoxin binding to the target cell andthe translocation of the light chain into the target cell) of thebotulinum toxin variant/analog shall be at most marginally (according tothe above definition), and preferably not at all, decreased and,secondly, that, in spite of its modification, the light chain istranslocated to its site of action, the cytosol of the motoneuron.

In contrast to the already aforementioned US 20020197278 the objectiveforming the basis of the present application does not aim to blockantigenic determinants, to decrease the antigenicity of the toxins or torestrict their diffusion away from the injection site.

The inventors of the present application surprisingly found, that thelight chain of botulinum toxins can be specifically PEGylated at itsN-terminus via insertion of at least one cysteine residue withoutsimultaneously impairing or even inhibiting the biological activity(according to the definition given above) of the botulinum toxins. Sucha PEGylated botulinum toxin is characterized by a surprisingly higher invivo stability (significant increase of HL and therewith an extended(pharmacological) duration of action).

The (therapeutic) duration of action of the natural botulinum toxins inthe patient depends on the serotype. Botulinum toxin type A ischaracterized by the longest duration of action of about 3 month. Theduration of action of botulinum toxin type C is of similar length asthat of type A, whereas botulinum toxin type B has a shorter duration ofaction. The effect of botulinum toxins type E and F lasts only about 2weeks in each case. The short duration of action of these two types doesnot allow their clinical application for the treatment of dystonia. Thepresent invention allows (1) the clinical application of all botulinumtoxins, even those having so far short-term activity, and (2) a moreadvantageous therapy with the already therapeutically utilized type Aand B toxins, as these need not be administered every three month, but,e.g., only every six month.

DESCRIPTION OF THE FIGURES AND SEQUENCES

FIG. 1: Summary of the oligonucleotides (SEQ ID NO:1 to 14) which havebeen employed in the cloning of the recombinant toxins and toxinfragments. Recognition sequences for restriction endonucleases areunderlined. The long sequences with the SEQ ID NOs: 16 and 15 showexamples for a recombinant (mutated) botulinum neurotoxin type A withattached cysteine residue N-terminal to a histidine tag (consisting of10 histidine residues) or a DNA that encodes it. The proline residue atthe N-terminus of the native toxins (position 1) that ismonocystronically expressed and translated was replaced by an alanineresidue to create a cleavage site in the multi-cloning site (MCS) of thevector. The vector comprises the coding sequence for the His-tagprecisely in the 5′-vicinity of this MCS. In addition a sequence whichis recognized by E. coil cells has already been inserted in place of thenative loop between the light and the heavy chain whereupon the nativepre-peptide (N-light chain-loop-heavy chain-C) is already cleaved intothe active two-chain neurotoxin without the addition of exogenousproteases and obtained as such during the recombinant production of theneurotoxin.

FIG. 2: Analysis of the PEGylation and control batches of C—H₁₀-BoNT/A(Example 5) in SDS polyacrylamide gels under non-reducing conditions.Lane 1: molecular weight marker; Lane 2: control batch; Lane 3:PEGylation batch.

DESCRIPTION OF THE INVENTION

In order to solve the stated object (see above) the inventors havedeveloped modified botulinum toxins. One aspect of the inventiontherefore relates to a modified botulinum toxin comprising a naturalheavy chain and a modified light chain, wherein the modification of thelight chain is that it comprises (1) an extension of the chain on itsN-terminus which has the following structure in the direction from theN- to the C-terminal end: -(C)_(n)-(tag)_(m)-(X)_(l)- , wherein

-   -   C represents a cysteine residue,    -   tag represents any tag, e.g. a Strep-tag or a His-tag, and    -   X represents the residue of any naturally occurring amino acid,    -   n represents an integer from 1 to 50,    -   m represents 0 or 1, and    -   l represents 0 or an integer from 1 to 50,        and (2) that at least one of the cysteine residues in the        extension of the chain is coupled to at least one chain of PEG.        Such a modified botulinum toxin is hereinafter also referred to        as PEGylated mutated botulinum or neurotoxin.

According to a preferred embodiment, the following conditions apply:

-   -   n=1, 2 or 3, m=0 or 1, l=0 or l≠0,    -   n=1, m=1, l=0; n=2, m=1, l=0; n=3, m=1, l=0;    -   n=1, m=0, l=0; n=2, m=0, l=0; n=3, m=0, l=0;    -   n=1, m=1, l≠0; n=2, m=1, l≠0; n=3, m=1, l≠0;    -   n=1, m=0, l≠0; n=2, m=0, l≠0; n=3, m=0, l‥0;        wherein the toxins per molecule are coupled to one, to two or to        three PEG molecules, depending on whether n=1, 2 or 3.

Thus preferably those modified botulinum toxins (especially of the typesA, B, and C1) fall within the aforementioned modified botulinum toxinsof the present invention whose light chain is modified in such a waythat it comprises an extension of the chain, wherein the extended chainhas one of the following sequences:

-(C)₁-(tag)₁-(X)₀-, -(C)₂-(tag)₁-(X)₀-,-(C)₃-(tag)₁-(X)₀-, -(C)₄-(tag)₁-(X)₀-, -(C)₅-(tag)₁-(X)₀-,-(C)₁-(tag)₁-(X)₁-, -(C)₂-(tag)₁-(X)₁-,-(C)₃-(tag)₁-(X)₁-, -(C)₄-(tag)₁-(X)₁-, -(C)₅-(tag)₁-(X)₁-,-(C)₁-(tag)₁-(X)₂-, -(C)₂-(tag)₁-(X)₂-,-(C)₃-(tag)₁-(X)₂-, -(C)₄-(tag)₁-(X)₂-, -(C)₅-(tag)₁-(X)₂-,-(C)₁-(tag)₁-(X)₃-, -(C)₂-(tag)₁-(X)₃-,-(C)₃-(tag)₁-(X)₃-, -(C)₄-(tag)₁-(X)₃-, -(C)₅-(tag)₁-(X)₃-,-(C)₁-(tag)₁-(X)₄-, -(C)₂-(tag)₁-(X)₄-,-(C)₃-(tag)₁-(X)₄-, -(C)₄-(tag)₁-(X)₄-, -(C)₅-(tag)₁-(X)₄-, etc.wherein in any of the above listed 25 preferred embodiments m can alsobe 0 instead of 1, and/or in each case all cysteine residues occurringin the extension of the light chain are also PEGylated.

Of course l can also be any integer from 11 to 50 or above 50, mostpreferred above 100 or above 250. However, the greater l is, the longerthe light chain becomes without the enzymatic/catalytic activity or the(total) biological activity (according to the above definition) beingcompromised by a specific upper limit for the length of the light chain.For reasons of practicability, however, an upper limit to l of 10-20 ispreferred so that preferred values for l are in the range of 1-10,unless l is 0, which is especially preferred.

Appropriate considerations also apply to n, wherein according to theinvention its upper limit has been set to 50 for practical andeconomical reasons. Preferably n is in the range of 1-10, more preferred1-5, so as not to insert, or not to have to insert, too many PEGmolecules (as all inserted cysteine residues are preferably PEGylated),and not to deprive the resulting PEGylated mutated botulinum/neurotoxinof its biological activity according to the aforementioned definition.This can easily occur, if too many cysteine and PEG residues areinserted or inserted at incorrect positions, as the light chain, perhapsdespite the binding of the toxin to the target cell, is not translocatedinto the target cell.

The structure of botulinum toxin type A was published by Lacy & Stevens1998 (Nat. Struct. Biol. 5, 898-902), the structure of botulinum toxintype B by Swaninathan & Eswaramoorthy (Nat. Struct. Biol 7, 693-699(2000)). Therefore the structure of the light chains is also known andone can determine which region of the heavy and the light chain,respectively are at the protein surface and thus may be suitable for acoupling with PEG. The frequently chosen procedure of binding anappropriately activated PEG (e.g., PEG-succinimidylpropionate) to thee-amino group of lysine residues did not seem promising to theinventors. An activated PEG can react with numerous lysine residues—evenin the binding region of the heavy chain—and this leads experimentallyto a drastic inactivation.

Instead, the inventors identified amino acid residues of the light chainwhich are suited initially to be replaced by at least one cysteineresidue and subsequently to be PEGylated on the at least one insertedcysteine residue. These modified botulinum toxins, hereinafter to becharacterized in more detail, which are also a preferred embodiment ofthe present invention and have as conjugates with PEG a sufficientbiological (including enzymatic/catalytic) activity (which by definitioncorresponds at least to 20%, preferably to 30-40%, to 50-70% or even to75-95% of the biological activity of the unmodified protein) withsimultaneously increased stability (in comparison to the correspondingnative neurotoxins) and will hereinafter also be referred to asPEGylated mutated botulinum or neurotoxins of the present invention.

These latter modified botulinum toxins of the present invention are alsoconjugates of mutated botulinum toxins with PEG. These modifiedbotulinum toxins are also coupled to PEG via separately insertedcysteine residues. For this purpose at least one, but optionally also 2,3, 4, 5, 10 or even all 20 of the first 20 amino acid residues of the Nterminal end of the light chain of the respective botulinum toxin is ineach case replaced by a cysteine residue. These modified botulinumtoxins also comprise a natural heavy chain and a modified light chain,wherein the modification of the light chain is such that at least one upto maximally 20 of the amino acid residues occurring naturally at the Nterminus are mutated to a cysteine residue. If applicable they comprisean additional N terminal extension, such that the sequence of the lightchain has the following structure in the direction from the N-terminusto the C-terminus: -(tag)_(m)-(X)_(l)-BoNT(X1-20C), wherein

-   -   C represents a cysteine residue,    -   tag represents any tag, e.g. a Strep-tag or a His-tag, and    -   X represents the residue of any naturally occurring amino acid,    -   m represents 0 or 1, and    -   l represents 0 or an integer from 1 to 50.

At least one of the maximally 20 cysteine residues at the N-terminus iscoupled to at least one chain of PEG.

Hence there result mutants for BoNT/A, which are characterized by atleast one—and not more than twenty—of the following replacements ofamino acid residues, such that a PEGylation can occur at the insertedcysteine residues. P1C, F2C, V3C, N4C, K5C, Q6C, F7C, N8C, Y9C, K10C,D11C, P12C, V13C, N14C, G15C, V16C, D17C, l18C, A19C, Y20C

According to a preferred embodiment, the following conditions apply:

-   -   m=1, l=0, only one out of the 20 N-terminal amino acid residues        is replaced by a cysteine residue, more preferable only the        residue at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m=0, l=0, only one out of the 20 N-terminal amino acid residues        is replaced by a cysteine residue, more preferable only the        residue at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m=1, l≠0, only one out of the 20 N-terminal amino acid residues        is replaced by a cysteine residue, more preferable only the        residue at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m=0, l≠0, only one out of the 20 N-terminal amino acid residues        is replaced by a cysteine residue, more preferable only the        residue at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m=1, l=0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 3, 1 and 4, 2 and 4, 1 and 5, 2        and 5, 3 and 5, 1 and 6, 2 and 6, 3 and 6, or 4 and 6;    -   m=0, l=0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 3, 1 and 4, 2 and 4, 1 and 5, 2        and 5, 3 and 5, 1 and 6, 2 and 6, 3 and 6, or 4 and 6;    -   m=1, l≠0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 3, 1 and 4, 2 and 4, 1 and 5, 2        and 5, 3 and 5, 1 and 6, 2 and 6, 3 and 6, or 4 and 6;    -   m =0, l≠0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 3, 1 and 4, 2 and 4, 1 and 5, 2        and 5, 3 and 5, 1 and 6, 2 and 6, 3 and 6, or 4 and 6;    -   m =1, l=0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, or        5 and 6;    -   m=0, l=0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, or        5 and 6;    -   m=1, l≠0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, or        5 and 6;    -   m=0, l≠0, only two out of the 20 N-terminal amino acid residues        are replaced by a cysteine residue, more preferable only the        residues at the positions 1 and 2, 2 and 3, 3 and 4, 4 and 5, or        5 and 6,        wherein the toxins per molecule are coupled to one or two PEG        molecules, depending on whether only one or two amino acid        residues have been replaced by (one) cysteine residue(s) at the        N-terminus.

Therefore preferably those botulinum toxins (especially of the types A,B, and C1) fall within the aforementioned modified botulinum toxinswhose light chain is modified in such a way that it comprises anextension of the chain at its N-terminus, wherein the N-terminus of theextended chain has one of the following sequences:

-(tag)₁-(X)₀-BoNT(P1C), -(tag)₁-(X)₀-BoNT(F2C), -(tag)₁-(X)₀-BoNT(V3C),-(tag)₁-(X)₀-BoNT(N4C), (tag)₁-(X)₀-BoNT(K5C), -(tag)₁-(X)₁-BoNT(P1C),-(tag)₁-(X)₁-BoNT(F2C), -(tag)₁-(X)₁-BoNT(V3C), -(tag)₁-(X)₁-BoNT(N4C),-(tag)₁-(X)₁-BoNT(K5C), -(tag)₁-(X)₂-BoNT(P1C), -(tag)₁-(X)₂-BoNT(F2C),-(tag)₁-(X)₂-BoNT(V3C), -(tag)₁-(X)₂-BoNT(N4C), -(tag)₁-(X)₂-BoNT(K5C),-(tag)₁-(X)₃-BoNT(P1C), -(tag)₁-(X)₃-BoNT(F2C), -(tag)₁-(X)₃-BoNT(V3C),-(tag)₁-(X)₃-BoNT(N4C), -(tag)₁-(X)₃-BoNT(K5C), -(tag)₁-(X)₄-BoNT(P1C),-(tag)₁-(X)₄-BoNT(F2C), -(tag)₁-(X)₄-BoNT(V3C), -(tag)₁-(X)₄-BoNT(N4C),-(tag)₁-(X)₄-BoNT(K5C), etc.wherein in any of the above listed 25 preferred embodiments m can alsobe 0 instead of 1, and/or in each case all N-terminally insertedcysteine residues are also PEGylated.

Of course, l can also be any integer from 11 to 50 or above 50, mostpreferred above 100 or above 250. However, the greater l, the longer thelight chain without the enzymatic/catalytic activity or the (total)biological activity of the neurotoxin becoming compromised with regardto the above mentioned definition by a specific upper limit for thelength of the light chain. For reasons of practicability, however, anupper limit of l of 10-20 is preferred, so that preferred values for lare in the range of 1-10, unless l is 0, which is especially preferred.

Unless explicitly specified otherwise, the following explanations applyto the modified botulinum toxins of the present invention, regardlesswhether it is the variant with inserted cysteine residue(s) in theN-terminal extension of the light chain or the variant with insertedcysteine residue(s) at the N-terminal end of the light chain.

Preferably, the modified botulinum toxin is a modified botulinum toxinderived from BoNT/A, BoNT/B, or BoNT/C1, but it also can be a botulinumtoxin of the types D, E, F, or G. It is also preferred that on the onehand all artificially inserted cysteine residues (preferably 1-10cysteine residues are inserted) comprise at least one PEG chain, but onthe other hand none of the naturally occurring cysteine residues of theheavy and light chains of the botulinum toxin is PEGylated.

It is self-explanatory to a person skilled in the art, that therelevance of the tag (m=1) resides in the easier purification of therecombinantly (e.g., in E. coli) produced modified botulinum toxin ofthe present invention. Thus, the tag is not used to increase thestability of the neurotoxin or its biological activity, but allows thesimplified and almost quantitative isolation of the modified botulinumtoxin from the bacterial culture.

Regarding the question of an appropriate choice of n (in case of thevariant with inserted cysteine residue(s) in the N-terminal extension ofthe light chain) or the number and position of the amino acid residuesto be replaced by cysteine residues (in case of the variant withinserted cysteine residue(s) at the N-terminal end of the light chain)one has to consider that preferably only as many cysteine residues areinserted as PEGylations are to be carried out (the same applies to thecase, where l≠0 and at least one amino acid residue X is a cysteineresidue). All of these inserted cysteine residues can preferably bePEGylated, and completely at that, without, and that is the surprisingfinding of the inventors, even one of the cysteine residues occurringnaturally in botulinum toxin being PEGylated on the heavy or on thelight chain (besides one intra- and intermolecular disulfide bridgeeach, botulinum toxin type A e.g. exhibits three additional cysteineresidues in the heavy chain (C₇₉₁, C₉₆₇, C₁₀₆₀) as well as twoadditional cysteine residues in the light chain (C₁₃₄ and C₁₆₅)). Inthis manner, a consistent (homogeneous) product in the form of aPEGylated mutated botulinum toxin can be obtained. Furthermore, it isreasonable to avoid too many cysteine or PEG residues being inserted orinserted at incorrect positions for an additional reason. This is sobecause (i) the toxin possibly may then become too bulky to bind to thetarget cell and/or (ii) the light chain is, perhaps despite binding ofthe toxin to the target cell, not sufficiently translocated into thetarget cell. In other words, the insertion of only one cysteine residuein this or that variant and its PEGylation routinely accomplishes thepurpose of the invention and is therefore especially preferred.

Accordingly, the PEGylated mutated botulinum/neurotoxins in accordancewith the present invention are, like the corresponding natural (native)botulinum/neurotoxins, biologically and enzymatically (i.e.,catalytically) active or exhibit in the sense of the definition givenabove not more than a marginally decreased biological andenzymatic/catalytic activity, but are more stable, partly evensubstantially more stable, than their natural precursor toxins fromwhich they were derived. Furthermore, a PEGylated mutated botulinumtoxin is preferred, whose PEGylated mutated (modified) light chainexhibits a higher stability in the cytosol of the motoneuron than theunmodified light chain of the corresponding native botulinum toxin.

As described above, the PEGylation of the light chain results in anincreased stability compared to the unmodified light chain. Inaccordance with the invention the PEG chain is attached to the lightchain thus, that, firstly, its enzymatic activity is unchanged or atmost marginally decreased (according to the definition given above),and, secondly, the modified light chain like the unmodified chain istranslocated into the cytosol of the motoneuron.

The His-tag as well as other tags, e.g., the Strep-tag, allows thestraightforward isolation of the mutated neurotoxin from the lysate oftransformed bacteria (e.g., E. coli). At the DNA level it is most simplyattached in 5′-direction to the coding region of the neurotoxin gene. Inthe case of the His-tag the isolation occurs by use of affinitychromatography on Ni-NTA-sepharose. In the next step, the mutatedneurotoxin that is isolated in this way is coupled to activated PEG. Aseries of activated PEG derivatives, e.g., PEG-maleimide,PEG-vinylsulfone, PEG-iodoacetamide, and PEG-orthopyridyl-disulfide areprovided by Nektar Therapeutics, and instructions for the PEGylation aresupplied. In accordance with the invention these PEG derivatives maycomprise different chain lengths: e.g., PEG derivatives with molecularweights of 5,000 Dalton, 10,000 Dalton, 20,000 Dalton, and 30,000 Daltonare commercially available and to be used in accordance with theinvention.

For the determination of the protease activity of the mutated andsubsequently PEGylated botulinum toxin the cleavage of the SNARE proteincorresponding to the serotype is quantitatively recorded. The activityis then compared to the activity of (i) the native neurotoxin (of thecorresponding serotype), (ii) the mutated neurotoxin with tag for thesimplified isolation and/or (iii) the mutated neurotoxin without tag.The activity of the mutated neurotoxin and of the PEGylated mutatedneurotoxin in accordance with the present invention is similar to theactivity of the native (non-mutated) neurotoxin (that is, the biologicalactivity, according to the definition above, of the mutated neurotoxinand of the modified neurotoxin according to the invention is at mostmarginally decreased in the sense of the definition given above comparedto the biological activity of the neurotoxin).

The total activity of the PEGylated neurotoxin is initially determinedin an ex-vivo model, the so-called diaphragm or hemidiaphragm-assay.Here the paralyzing activity is determined on a nerve-musclepreparation. In accordance with the invention the biological activity ofthe modified as well as of the mutated and subsequently PEGylatedneurotoxin is at least 20%, preferably 30-40% or 50-70% or even 75-95%of the biological activity of the unmodified (native) protein(biological activity is also to be understood according to thedefinition given above).

The toxicity of the PEGylated mutated neurotoxin according to theinvention can be tested in the mouse LD₅₀-assay, whereby the dose isbeing determined that after i.p. application is lethal for half the micefrom a group.

The duration of action of the PEGylated mutated neurotoxin according tothe invention is determined in vivo, also with a mouse. The periodduring which the muscle remains paralyzed is determined after injectionof a sublethal dose of a PEGylated mutated botulinum toxin type Aaccording to the present invention or of the corresponding nativebotulinum toxin in the gastrocnemius muscle of the hindpaw. Theparalysis potency is classified by way of a chart. The duration ofaction, which is shorter in the mouse than in men, is extended by30-150% depending on the modified botulinum toxin type A used (measuredin comparison to the non-PEGylated and unmutated botulinum toxin typeA).

The PEGylated mutated botulinum toxin according to the present inventioncan be processed in an appropriate formulation to a finished drugproduct, which includes a dose (or an integral multiple of the dose) inthe range of the therapeutic dose (e.g., 100 LD₅₀-units per injectionvial). According to a preferred embodiment the pharmaceuticalcomposition is stabilized without addition of human serum albumin (HSA).However, it may also be stabilized with human serum albumin (HSA). Inthis regard the use of a HSA-free composition for the stabilization ofPEGylated active protein substances as described in WO 2005/007185 isespecially preferred. In accordance with a further preferred embodimentthe pharmaceutical composition of the present invention is inlyophilized form or fluid. Both forms are suitable, optionally afteruptake in an appropriate solvent, for i.m. injection in the muscle to betreated.

The PEGylated mutated botulinum toxin with greater stability and halflife or the pharmaceutical exhibiting these can be used for therapy ofvarious dystonia as spasmodic dystonia, laryngeal dystonia, cervicaldystonia, focal hand dystonia, blepharospasm, strabism, cerebralparesis, hemifacial spasms, spasticity, spasmodic colitis, anismus,TICS, tremors, bruxism, anal fissure, achalasia, dysphagia,hyperhidrosis as well as for removal of facial wrinkles.

Additional aspects of the present investigation relate to (1) a nucleicacid, which encodes the above explicitly described modified botulinumtoxin with increased stability (especially the nucleic acid is DNA); (2)a vector, comprising the nucleic acid according to (1); and (3) a hostcell, comprising the vector according to (2) (especially the host cellis a prokaryotic, in particular, an E. coli host cell).

The following examples illustrate the invention in detail withoutlimiting the invention to the aforementioned specific parameters.

EXAMPLES Example 1 Cloning and Expression of Botulinum Neurotoxin Type A(BoNT/A)

For cloning the DNA sequences of the light chain as well as of thetranslocation domain, chromosomal DNA was isolated from a culture ofClostridium botulinum type A (strain ATCC 3502). A coding gene fragmentwith modified loop sequence for the light chain of BoNT/A was obtainedby using PCR amplification with the primers SEQ ID NO:1 and SEQ ID NO:2.The PCR amplificate was cloned via the restriction cleavage sites forNco I and Bgl II into the expression plasmid pQE-H₁₀, which was derivedfrom pQE-60 and encodes a His-tag (consisting of 10 histidine residues)at the 5′-end of the cloning site. The plasmid pQE-H₁₀-BoNT/A-L wasgenerated by this cloning method. A coding gene fragment for the heavychain of BoNT/A was obtained by using PCR amplification with the primersSEQ ID NO:3 and SEQ ID NO:4. It was cloned by means of the restrictioncleavage sites for Stu I and Bgl II to the 3′-end of the loop sequenceof the light chain in pQE-H₁₀-BoNT/A-L (plasmid pQE-H₁₀-BoNT/A). The E.coli expression strain M15[pREP4] (Qiagen) was transformed with theplasmid pQE-H₁₀-BoNT/A. The expression of the recombinant toxins wasrealized by a graded induction with 500 μM IPTG (final concentration) at25° C. over night. The cells were solubilized through lysozyme andultrasound treatment in a 50 mM phosphate buffer at pH 8.0 with 300 mMNaCl. The centrifuged lysate was incubated for 5 h at room temperatureand after intermittent storage at −20° C. chromatographed on a Ni-NTAagarose column. Finally the elution fractions were dialyzed against acoupling buffer (100 mM sodium dihydrogen phosphate pH 7.5, 150 mM NaCl,10 mM EDTA) and the protein concentration was determined. An analysis onSDS polyacrylamide gel showed that under reducing conditions two strongbands at about 50 kDa and 100 kDa as well as a weak band at 150 kDa werestained with Coomassie whereas under non-reducing conditions only oneband was observed at about 150 kDa, which corresponds to the bandingpattern of a botulinum neurotoxin type A in its prevailing two-chain,disulfide-bridged structure.

Example 2 Cloning and Expression of Botulinum Neurotoxin Type B (BoNT/B)

Cloning and expression of a botulinum neurotoxin type B equipped with ahistidine tag (consisting of 10 histidine residues) at the N-terminuswas carried out analogously to the type A toxin. Chromosomal DNA fromClostridium botulinum type B (strain Okra) as well as the primer SEQ IDNO:5 and SEQ ID NO:6 or SEQ ID NO:7 and SEQ ID NO:8 were employed forthe amplification of the light and heavy chains.

Example 3 Cloning and Expression of Botulinum Neurotoxin Type C1(BoNT/C1)

Cloning and expression of a botulinum neurotoxin type C1 equipped with ahistidine tag (consisting of 10 histidine residues) at the N-terminuswas done analogously to the type A toxin. Chromosomal DNA fromClostridium botulinum type C (strain 205) as well as the primer SEQ IDNO:9 and SEQ ID NO:10 or SEQ ID NO:11 and SEQ ID NO:12 were employed forthe amplification of the light and heavy chain.

Example 4 Cloning and Expression of C—H₁₀-BoNT/A

To allow for a N-terminal PEGylation a cysteine residue was attached tothe amino acid sequence N-terminal to the histidine tag (consisting of10 histidine residues). This was achieved through site-directedmutagenesis in the sequence region of pQE-H₁₀-BoNT/A, which encodes theHis-tag. The QuickChange Site Directed Mutagenesis Kit of Stratagene wasemployed. The mutagenesis reaction was carried out with the primers SEQID NO:13 and SEQ ID NO:14. The nucleotide exchange in the DNA sequencewas verified by DNA sequencing of the isolated clones. The expressionand the purification of the mutated toxin were carried out analogouslyto Example 1.

Example 5 PEGylation of C—H₁₀-BoNT/A

1.2 mg of C—H₁₀-BoNT/A was incubated for 30 minutes in 1 mM DTT toreduce the disulfide-bridged dimers. For separation of the reducingagent a buffer exchange to coupling buffer was carried out on a PD-10column. The toxin solution was concentrated to 3.6 mg/ml by means ofultrafiltration. A small aliquot was incubated untreated as controlsample, the remainder of the solution was mixed with a 5-fold molarexcess of mPEG-Mal-5000 (Nektar Therapeutics) and rotated at ambienttemperature over night. In order to avoid derivatization reactions onfurther cysteine residues during the sample preparation for SDS-PAGE,the PEGylation reagent was saturated with a 5-fold excess of L-cysteine.The SDS gel showed a strong additional band with slightly reducedmobility in comparison to the control batch under non-reducingconditions, while the intensity of the original toxin band at 150 kDawas significantly decreased (FIG. 2).

Example 6 In Vitro-Activity Assay

Determination of the specific protease activity (that is, the catalyticactivity without binding or translocation) of the PEGylated mutatedBoNT/A derivative was done in the ELISA format. For this purpose, arecombinant polypeptide was cloned, consisting in the N-terminal regionof the common fusion partner glutathion-S-transferase (GST) and of aC-terminal peptide sequence, which comprises the C-terminal 17 aminoacid residues of SNAP 25. These 17 amino acid residues represent theregion of the substrate protein SNAP 25, in which BoNT/A specificallycleaves. After coating of a microtiter plate with the fusion proteinincubation was done with H₁₀-BoNT/A as reference sample or with themutants in their PEGylated and non-PEGylated form. The detection of thecleavage products generated in each case was done with an antibody,which specifically detects the newly formed C-terminus. The values forC—H₁₀-BoNT/A in its non-PEGylated as well as PEGylated form and forBoNT/A (reference sample) are listed in Table 3. Considering thevariation limit of the assay one can observe that through theintroduction of the mutation and the subsequent PEGylation the catalyticactivity of the botulinum toxin was not decreased, but instead wasenhanced.

TABLE 3 Relative activity [%] H₁₀-BoNT/A 100 C—H₁₀-BoNT/A 100mPEG-C—H₁₀-BoNT/A 121.1

The specific activity was determined in protease units/ng protein.

Example 7 Determination of the Ex-vivo Activity in the HemidiaphragmAssay

For the determination of the total activity of the toxin derivatives,i.e., the binding of the modified neurotoxin to the receptor of thetarget cells and the translocation into the nerve cell and proteolysisof the SNARE substrate, the paralysis time of a nerve-muscle preparationfrom mouse was determined after intoxication. Again H₁₀-BoNT/A served asa reference sample. The values of the relative activity are listed inTable 4. The reduction in the activity of the modified botulinum toxinsaccording to the present invention to 20-30% as compared to thereference sample is obviously based on the reduced ability of the toxinto bind to the target cells and on the reduced ability to translocatethe toxin into the target cells.

TABLE 4 Relative activity [%] H₁₀-BoNT/A 100 C—H₁₀-BoNT/A 22.5mPEG-C—H₁₀-BoNT/A 30

Example 8 Duration of Action of PEGylated Botulinum Toxin Type A

The duration of action of PEGylated mutated botulinum toxin(mPEG-C-H₁₀-BoNT/A) was assayed with CD 1-mice. 10 mice each receivedi.m. injections (2×0.05 mL) of (i) mutated, (ii) PEGylated mutated or(iii) native botulinum toxin in a dosage of 0.4 or 0.6 LD₅₀-units/mouse(in physiological saline+1 mg/mL HSA) into the gastrocnemius muscle ofthe hindpaw. Afterwards the paralysis of the muscle was evaluated on adaily basis by using a chart (minimal, gentle, severe paralysis). Nomore paralysis of the muscle was observed after 25 days in the animalstreated with the native neurotoxin. In the animals treated with themutated or PEGylated mutated botulinum toxin, the duration of action wasextended for 7-20 days.

1. A method of treating dystonia or for removing facial wrinklescomprising treating a subject with modified botulinum toxin comprising anatural heavy chain and a modified light chain, characterized in thatthe modification of the light chain comprises (i) at least one aminoacid residue, and maximally 20 amino acid residues, of the amino acidresidues occurring naturally at its N-terminus mutated to a cysteineresidue, wherein at least one of the maximally 20 cysteine residues atthe N-terminus is coupled to at least one chain of polyethylene glycol(PEG).
 2. The method according to claim 1, wherein the dystonia is aspasmodic dysphonia, a laryngeal dystonia, a cervical dystonia, a focalhand dystonia, a blepharospasm, a strabism, a cerebral paresis, ahemifacial spasm, spasticity, a spasmodic colitis, anismus, TICS,tremor, bruxism, an anal fissure, an achalasia, a dysphagia, or ahyperhidrosis.